Hardy Diagnostics HUGO

PHOSPHATE BUFFERED SALINE (PBS) WITH TWEEN®

Cat. no. R199 PBS with Tween®, 1ml 20 tubes/box
Cat. no. U199 PBS with Tween®, 500ml Polycarbonate Bottle, 500ml 1 bottle/box

INTENDED USE

Hardy Diagnostics Phosphate Buffered Saline (PBS) with 0.3% Tween® 20 is used during specimen processing for recovery of bacterial spores. It is recommended to be used specifically for environmental sampling.

This product is not intended to be used for the diagnosis of human disease.

SUMMARY

Commonly used in biological and biomedical research, Phosphate Buffered Saline (PBS) has many applications due to its isotonic nature. PBS can be used as a diluting agent in preparing decimal dilutions and as a rinsing agent for rinsing labware containing cells. Phosphate salts are nontoxic to living cells and have a high buffering capacity; pH maintenance is important in retaining cell viability and in the recovery and revitalization of injured or damaged cells during bacterial culture and analysis. Phosphate Buffered Saline is generally used as a 10mM working solution containing a mixture of sodium chloride and inorganic phosphate salts.

Phosphate Buffered Saline is recommended as a rinsing or diluting fluid by the Food and Drug Administration in the Bacteriological Analytical Manual (BAM), by the Association of Analytical Communities (AOAC) in the Official Methods of Analysis, by the United States Pharmacopeia in the USP-NF, and by the American Public Health Association (APHA) in the Compendium of Methods for the Microbiological Examination of Foods and Standard Methods for the Examination of Water and Wastewater.(1-3,8,9) PBS is also recommended in the Clinical Microbiology Procedures Handbook and other laboratory texts for diluting or rinsing specimens for microbiological, histological, immunological or molecular testing.(4-7,10)

FORMULA

10 millimolar solution of Phosphate Buffered Saline with 0.3% Tween® 20.

Final pH 7.2 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-30ºC. away from direct light. Products should not be used if there are any signs of deterioration (discoloration), contamination, or if the expiration date has passed.

PRECAUTIONS

PROCEDURE

Consult listed references for information on specimen collection and specific standard methods.(1-5)

General Dilution Guidelines:

1:10 Serial Dilutions
1. Using a sterile pipet, aliquot 10mL of test suspension to 90mL of PBS diluent. Mix thoroughly. This yields a 1:10 dilution.

2. Use a second sterile pipet to aliquot 10mL of the 1:10 dilution prepared in step 1 into a second 90mL filled vessel of PBS diluent. Mix thoroughly. This yields a 1:100 dilution.

3. Continue aliquoting 10mL of subsequent dilutions into 90ml filled PBS diluent vessels until the desired concentration of test sample is obtained. Each succeeding dilution increases by a factor of 10. A separate sterile pipet should be used with each dilution.

1:100 Serial Dilutions
1a. Using a sterile pipet, aliquot 1ml of test suspension to 99ml of PBS diluent. Mix thoroughly. This yields a 1:100 dilution.

2a. Use a second sterile pipet to aliquot 1ml of the 1:100 dilution prepared in step 1a into a second 99mL filled vessel of PBS diluent. Mix thoroughly. This yields a 1:10,000 dilution.

3a. Continue aliquoting 1mL of subsequent dilutions into 99mL filled PBS diluent vessels until the desired concentration of test sample is obtained. Each succeeding dilution increases by a factor of 100. A separate sterile pipet should be used with each dilution.

Note: Hardy Diagnostics PBS with Tween® is filled into a 15x120mm polypropylene centrifuge tube. The durable tube reduces the chance of exposure to potentially infectious spores in the sample. It is recommended that specimens processed in tis centrifuge tube be heat-shocked for 45-50 minutes at 67ºC. to effectively kill the vegetative cells.(11) The time and temperature for processing the sample in the centrifuge tube has been modified from the original protocol (65ºC. for 30 minutes) because the polypropylene tube has greater insulation properties inhibiting heat transfer.(6) Data to support this modified protocol is available upon request.(11)

INTERPRETATION OF RESULTS

See listed references for additional information.

LIMITATIONS

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, inoculation spreaders, pipet tips, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Bacillus subtilis
ATCC® 6633
**
on A10
18-24hr 35°C Aerobic Growth
Bacillus subtilis
ATCC® 6633
**
in R20
18-24hr 35°C Aerobic Growth

**Inoculate PBS with Tween® with the test organism. Refer to Procedure Section above.

User Quality Control

PHYSICAL APPEARANCE

Phosphate Buffered Saline (PBS) with Tween® should appear clear and colorless, with no precipitate or debris.

REFERENCES

1. American Public Health Association. Standard Methods for the Examination of Water and Wastewater. APHA, Washington, D.C.

2. American Public Health Association Technical Committee on Microbiological Methods for Foods. Compendium of Methods for the Microbiological Examination of Foods, APHA, Washington, D.C.

3. Association of Official Analytical Communities. Official Methods of Analysis, AOAC, Washington, D.C.

4. Bullock, G.R. and Petrusz, P. 1989. Techniques in Immunocytochemistry, Volumes 1, 2, 3 and 4, Academic Press.

5. Cseke, L.J., P.B. Kaufman, G.K. Podila, and C.J. Tsai. 2004. Handbook of Molecular and Cellular Methods in Biology and Medicine. CRC Press. Taylor & Francis LLC. Boca Raton, FL.

6. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

7. Jorgensen et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.

8. The Official Compendia of Standards. 2008. USP27-NF22. United States Pharmacopeial Convention, Rockville, MD.

9. U.S. Food and Drug Administration. Bacteriological Analytical Manual. AOAC, Arlington, VA. www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/default.htm

10. Walker, J.M. 1984. Methods in Molecular Biology. The Humana Press Inc. Clifton, NJ.

ATCC is a registered trademark of the American Type Culture Collection.

Tween is a registered trademark of ICI Americas, Inc.

062916vr